The innate immune system employs three general strategies for self/nonself discrimination: recognition of "missing self," recognition of "induced self," and recognition of "microbial nonself." The goal of this proposal is to understand the structural basis for missing self recognition by natural killer (NK) cell receptors, and for microbial nonself recognition by peptidoglycan recognition proteins (PGRPs). NK cell function is regulated by a dynamic balance between activating receptors that trigger NK cytolytic activity and inhibitory receptors that detect the lack of normal MHC class I (MHC-I) expression (missing self). PGRPs are pattern recognition receptors which bind peptidoglycans (PGNs), a unique component of bacterial cell walls (microbial nonself). The specific objectives of the proposed studies are: 1) Determination of the crystal structures of selected mouse Ly49 NK receptors bound to their MHC-I ligands in order to define the different strategies these receptors have evolved to recognize missing self. These structures should resolve whether the variable binding modes we observed in the Ly49C/H-2Kb and Ly49A/H-2Dd complexes represent distinct intermediates in a sequential binding pathway common to all Ly49s, or whether different Ly49s engage MHC-I in topologically distinct orientations. Crystal structures will be correlated with the affinity and stoichiometry of Ly49/MHC-I interactions in solution. 2) Determination of the structures of Ly49s in free form in order to establish whether conformational changes in these homodimeric receptors, either in subunit association or the MHC-binding site, occur upon ligand engagement. 3) Definition of the affinity parameters governing Ly49 inhibitory activity. The functional activity of affinity-matured Ly49 receptors from yeast display libraries will be examined using a T cell transfection system in which the TCR serves as the activating receptor and Ly49 variants provide negative regulatory signals. 4) Determination of the crystal structure of the key activating NK receptor 2B4 (CD244) bound to its CD48 ligand. 5) Determination of the structural basis for MHC class II recognition by Lag-3 (CD223), a CD4-like molecule implicated in NK-mediated cytotoxicity that, in marked contrast to CD4, forms stable dimers in solution. 6) Determination of the structural basis for PGN recognition by human PGRPs. To this end, we have undertaken a structure determination of the PGN-binding domain of PGRP-Ialpha. These studies will be extended to include human PGRP-Ibeta, PGRP-S and PGRP-L, as well as complexes with synthetic analogs of PGNs.